PROJECT SUMMARY The objective of this application is to determine how tissue nonspecific alkaline phosphatase (TNAP) enzymatic activity preserves blood-brain barrier (BBB) function in ischemic stroke. Increased BBB permeability and oxidative stress are two potential mechanisms through which cerebral ischemia and reperfusion injury elicit BBB dysfunction and subsequent functional deficits in acute ischemic stroke. TNAP is a highly enriched enzyme in cerebral microvessels whose function in brain microvascular endothelial cells (BMECs) is poorly understood. Our preliminary data generated in a cellular BBB model of ischemia-reperfusion injury demonstrate that TNAP activity stimulates a novel mechanism which enhances cAMP-mediated signaling pathways that suppress Rho kinase (ROCK) activity and preserve BBB function. These intriguing findings led us to propose the novel concept that, in the face of cerebral ischemic injury, TNAP-ROCK signaling mechanisms integrate systemic signals at the BBB to sustain cerebral function and protect against post-stroke behavioral deficits. To address this novel concept, the application will investigate the central hypothesis that protection from ischemic stroke-induced impairment of BBB function is mediated by brain endothelial cell TNAP and its associated signaling cascades. The proposed studies will test this hypothesis in a mouse model of experimental ischemic stroke, transient middle cerebral artery occlusion (tMCAO), and in an ex vivo model of ischemia-reperfusion injury, oxygen-glucose deprivation (OGD). The experimental design will employ a mouse model with conditional deletion of TNAP in endothelial cells (VE-cKO) and its wild type littermates to interrogate TNAP?s effects on BBB permeability, short- term functional recovery, and BMEC signaling pathways post-stroke. Aim 1 will elucidate how TNAP preserves BBB permeability and promotes barrier function during cerebral ischemia and reperfusion injury. Aim 2 will interrogate the role of TNAP-ROCK signaling mechanisms in an OGD model of injury. Aim 3 will integrate the experiments in Aims 1 and 2 to prepare a competitive R01 application that will lead to independence of COBRE funding. Taken together, the studies in this proposal will uncover novel insights into the mechanisms that link TNAP enzyme activity to BBB integrity, barrier function, short-term functional recovery in ischemic stroke. The translational impact of these studies may uncover TNAP and its associated signaling cascades as novel therapeutic targets in ischemic stroke and other cerebrovascular disorders.